This invention is in the field of bacterial infections and is more particularly concerned with infection by Pseudomonas aeruginosa and is specifically concerned with enzymes involved with the synthesis of O antigens, namely WbpP and methods for the use and assay for WbpP.
Pseudomonas aeruginosa is an opportunistic gram-negative bacterium that can cause life-threatening infections in patients with cystic fibrosis or burn wounds (Hancock et al. (1983)). It produces a wide variety of virulence factors such as proteases, toxins, alginate and lipopolysaccharides (LPS) (Hancock et al. (1983)). Two forms of LPS have been identified: the antigenically conserved A-band LPS, and the variable O-antigen or B-band. B-band LPS is particularly important in the initial steps of the infection, and particularly for evasion of host defenses and colonization (Cryz et al. (1984); Pier et al. (1982)). It contributes to causing initial tissue damage and inflammatory responses in the lungs of patients with cystic fibrosis (Cryz et al. (1984)). P. aeruginosa mutants deficient in B-band LPS biosynthesis are more sensitive to serum killing (Hancock et al. (1983); Schiller et al. (1983); Goldberg et al. (1996)) and are more susceptible to phagocytosis (Engles et al (1985)) than wild-type bacteria. They are found almost avirulent in mouse models (Cryz Ct al. (1984)). B-band LPS is the basis for classification of P. aeruginosa in 20 different serotypes. Among these, serotypes O6 and O11 are the most clinically relevant in epidemiological studies (Pitt (1989)). To date, the prognosis for a cystic fibrosis patient infected with either serotype of P. aeruginosa is rather poor due to intrinsic multidrug resistance of P. aeruginosa. Such resistance is due partly to a highly impermeable outer membrane and partly to the presence of multidrug efflux pumps (Poole et al. (1993); Pool et al. (1996); Srikurnar et al. (1999)). Hence, B-band LPS biosynthesis has become an important target for drug discovery.
The genetics of B-band LPS biosynthesis are well documented in serotypes O5, O6 and O11 (Burrows et al. (1996); Belanger et al. (1999); Dean et al. (1999)) and were thoroughly reviewed recently (Rocchetta et al. (1999)). For each of these scrotypes, the entire cluster of genes responsible for B-band LPS synthesis has been sequenced and putative pathways for the synthesis of the corresponding O-antigens have been proposed based on homology studies. In serotype O11, the functional role of these genes awaits further studies. However, in serotypes O5 and O6, extensive functional characterisation has been performed by knockout construction and complementation analysis, using not only genes from P. aeruginosa but also homologues found in other organisms. Despite these efforts, ambiguities persist that can only be alleviated by direct biochemical characterisation of the proteins involved. Such a characterisation will also allow screening for inhibitors that might be useful for therapeutic purposes, especially if performed for enzymes found in the clinically relevant serotype 06.
The present inventors have cloned the nucleic acid sequence of WbpP in an expression vector that allows the production for the first time of large amounts of the WbpP protein.
Accordingly, in one embodiment, the present invention provides an isolated nucleic acid molecule comprising:
(a) a nucleic acid sequence as shown in FIG. 9 (SEQ.ID.NO.:1), wherein T can also be U;
(b) nucleic acid sequences complementary to (a);
(c) nucleic acid sequences which are homologous to (a) or (b);
(d) a fragment of (a) to (c) that is at least 15 bases, preferably 20 to 30 bases, and which will hybridize to (a) to (d) under stringent hybridization conditions, or
(e) a nucleic acid molecule differing from any of the nucleic acids of (a) to (c) in cod on sequences due to the degeneracy of the genetic code.
according to another embodiment the present invention provides an isolated nucleic acid molecule having the sequence shown in FIG. 9 (SEQ.ID.NO.:1) (or variants or fragments thereof. The present invention also provides a protein encoded by the nucleic acid sequence of FIG. 9 (SEQ.ID.NO.:1) and shown in FIG. 10 (SEQ.ID.NO.:2). The protein possesses a N-terminal extension as a luistidine tag that allows fast and efficient purification of the enzyme.
Having isolated and purified a WbpP enzyme has allowed the inventors to characterize its function. The O-antigen of B-band LrS of serotype O6 consists of a tetrasaccharide repeat of xe2x86x92-xcex1-D-3 O-acetyl, 6 amino-GalNAcA-(1xe2x86x924)-xcex1-D-6-amino-GalNFmA-(1xe2x86x923)-xcex1-D-QuiNAc-(1xe2x86x922)-xcex1-L-Rha-(1xe2x86x92(15-17). GalNAcA is thought to be synthesized in vivo via epimerisation and dehydrogenation of UP-GIcNAc, the main precursor of surface-associated carbohydrate synthesis (Belanger et al. (1999); Kochetkov et al. (1973); Virlogeux et al. (1995)). The product of the epimerisation reaction, UDP-GalNAc, is an important intermediate for the synthesis of polysaccharide structures that contain GalNAcA or a derivative, not only in P. aeruginosa but also in other orgamisms. The gene wbpP is part of the B-band LPS cluster in P. aeruginosa O6 (Belanger et al. (1999)). The amino acid sequence of WbpP (FIG. 10 (SEQ ID NO.: 2)) shows 23% identity with the C4 UDP-Glc epimerase GalE from Escherichia coli. It also shows 66% identity with WcdB, an enzyme thought to be involved in the formation of GalNAcA residues present in the Vi polysaccharide of Salmonella typhi (Virlogeux et al. (1995)). Disruption of the wbpP gene in a knockout mutant results in loss of B-band LPS production in P. aeruginosa and, this deficiency is fully alleviated after complementation by the wcdB homologue (Belanger et al. (1999)). Though no biochemical evidence is available for either WbpP or WcdB, sequence comparisons with other proteins and carbohydrate composition analysis suggest that they are C4 epimerases that transform UDP-GlcNAc into UDP-GalNAc in vivo.
A functional assignment relying mainly on homology studies is particularly problematic in the case of putative epimerases. Epimerases belong to the short-chain dehydrogenase/reductase (SDR) enzyme family. This family includes enzymes responsible for a wide variety of functions (Jornvall et al. (1995); Jornvall (Adv. Exp. Med. Biol. 463, 359-364 (1999)); Jornvall et al. (PEBS Lett. 445, 261-264 (1999)). Most of these enzymes possess common features which include the presence of the G-x-x-G-x-x-G signature for nucleotide binding proteins and the presence of alternating xcex1 and xcex2 structures which delineate a typical nucleotide binding Rossman fold at their N-terminus (Rossmann, et al. (1975); Bauer et al. (1992)). Moreover, they share a conserved catalytic triad S-(x)24--Y-(x)33-K probably involved in initiation of the catalytic process. All these features arc present in WbpP and they match perfectly with those found in the C4 UDP-Gal epimerase GalE found in E. coli (FIG. 1) but also those of other enzymes with different functions such as RFPG, a dTDP-glucose 4,6-dehydratase present in E. coli (Marolda et al. (1995)). Here is described the work conducted by the inventors to perform the biochemical analysis necessary to prove without ambiguity the function of WbpP, namely, that of a C4 UDP-GlcNAc epimerase. This describes the first epimerase for the N-acetylated form of the substrate.
The present invention also includes expression vectors containing the nucleic acid molecules of the present invention. The expression vectors will contain the necessary regulatory regions to provide for expression of the histidine tagged protein.
The present invention further provides host cells which have been transformed with the expression vectors of the present invention.
Accordingly, the present invention provides a method for expressing a protein having WbpP comprising inserting a nucleic acid molecule encoding the protein into an appropriate expression vector; transforming a host cell with the expression vector; and providing conditions which allow for expression of the protein. Preferably the protein is expressed in soluble and active form.
In another embodiment the present invention provides a method of assaying for WbpP activity in a sample comprising adding a sufficient amount of UDP GalNAc to the sample, under appropriate conditions for reaction, and assaying for UDP GlcNAc, wherein the appearance of UDP GlcNAc reflects the presence of WbpP activity. Preferably the amount of UDP GlcNAc which appears is determined, and preferably the amount of UDP GlcNAc which is determined is correlated to the amount of the substance providing the WbpP activity in order to determine the amount of the substance providing the WbpP activity which is in the sample. Preferably the amount of UDP-GlcNAc formed is determined by spectropholometric assay using p-dimethylaminobenzaldehyde (DMAB).
According to another embodiment, the present invention provides a method of assaying for WbpP activity in a sample comprising adding a sufficient amount of UDP GlcNAc to the sample, under appropriate conditions for reaction, and assaying for changes in the presence of UDP GlcNAc, wherein a disappearance, or reduction in UDP GlcNAc reflects the presence of WbpP activity. Preferably changes in the amount of UDP GlcNAc are determined and preferably the amount of UDP-GlcNAc which is determined is correlated to the amount of the substance providing the WbpP activity in order to determine the amount of the substance providing the WbpP activity which is in the sample. Preferably the amount of UDP-GlcNAc is determined by spectrophotometric assay using p-dimethylaminobenzaldeliyde (DMAB).
In another aspect, the present invention provides an assay for detecting inhibitors of a substance with WbpP activity. Accordingly, the present invention further provides a method for screening for an inhibitor of a substance with WbpP activity comprising (a) incubating a test sample containing (i) an substance with WbpP activity, (ii) a substance suspected of being an inhibitor of the substance; and (iii) UDP-GlcNAc or UDP-GalNAc; (b) stopping the reaction; (c) comparing the amount of UDP-GlcNAc, or UDP-GalNAc in the test sample with the amount in a control sample (that does not contain the substance suspected of being an inhibitor) wherein a decrease in the amount of GlcNAc, or UDP-GalNAc in the control sample as compared to the test sample indicates that the substance is an inhibitor of the substance with WbpP activity.
The present invention further provides a method for diagnosing or detecting an infection, preferably those associated with Pseudomonas aeruginosa, comprising detecting the presence of a nucleic acid or protein of the present invention in a biological sample.
The present invention also provides a method for inhibiting infection of an animal, preferably those infections associated with Pseudomonas aeruginosa, comprising inhibiting the transcription or translation (i.e., expression) of a nucleic acid molecule of the present invention. The expression of the nucleic acid molecule may be inhibited using antisense oligonucleotides that are complimentary to the nucleic acid molecules of the invention.
The present invention further provides a method for inhibiting infection in an animal comprising inhibiting the activity of the proteins of the present invention. The proteins of the present invention may be inhibited by using an antibody that is specific for the protein.
According, to another aspect, the present invention provides a method for converting UDP-GlcNAc to UDP-GalNAc. UDP-N-acetylgalactosamine (UDPGalNAc) may be used as a substrate in an assay. UDP-GalNAc is very expensive. Consequently, the inventors have developed a method of producing UDPGalNAc from UDP-N-acetylglucosamine (UDPGlcNAc) which is less costly: Namely the present invention provides a method of producing UDPGalNAc comprising incubating an epimerase in the presence of UDPGlcNAc under appropriate conditions for the production of UDPGalNAc. Preferably, the epimerase is WbpP from serotype O6 (WbpPO6). Preferably the amount of UDP-GalNAc formed is determined by spectrophotometric assay using p-dimethylaminobenzaldehyde (DMAB).
The present invention also provides a method of inhibiting the epimerization of UDP-GalNAc. Preferably inhibition may be achieved through inhibition of expression of the nucleic acid molecule using antisense oligonucleotides that are complimentary to the nucleic acid molecules of the invention.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.